Lung cancer, the leading cause of cancer death in the United States, is often detected in late stages of its development. A better understanding of the mechanisms by which lung cancer initiates and progresses has a great potential for the development of new methods for reducing lung cancer mortality. There is, however, a lack of understanding of events occurring during the early stages of lung tumorigenesis and particularly the cell types in which deleterious events accumulate. Studies of lung injury and regeneration have led this group to identify tissue stem cells, termed variant Clara cells (vCE), that are sequestered within specialized microenvironments defined by the broncheoalveolar duct junction (BADJs) and neuroepithelial bodies (NEBs) in bronchioles. It remains unclear, however, whether these stem cells are susceptible to the type of carcinogen-induced mutations in the K-ras oncogene which have been identified in a high proportion of lung tumors from both lung cancer patients and from carcinogen-treated mice. The main aim of this proposal is to gain insight into molecular events occurring during the early stages of lung tumorigenesis. Specifically, we hypothesize that K-ras mutations occur in stem cells, such as the vCE or the recently identified bronchioalveolar stem cell (BASC). For this purpose, we propose to compare the frequencies and types of K-ras mutations in vCE and BASC with that in non-stem cells. K-ras mutations in cells from control mice and those treated with NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone], a potent lung carcinogen found in tobacco smoke will be compared at various time points after chemical-treatment. Furthermore, we will test the hypothesis that treatment of mice with NNK, followed by naphthalene, an agent that activates stem cells secondary to non-stem cell depletion, provides an approach for identifying stem cells as the cells of origin of K- ras mutations in stem cell niches containing K-ras mutations, and for assessing the impact of these mutations on stem cell self-renewal and differentiation. PUBLIC HEALTH RELEVANCE: Lung cancer, the leading cause of death in the United States, is often detected in late stages of its development. A better understanding of the mechanisms by which lung cancer initiates and progresses provides a great potential for the development of methods for reducing lung cancer mortality.